This is an amended R01 renewal application, the purpose of which is to study the molecular mechanisms of the rapid regulation of the type-1 sodium-proton exchanger, NHE-1 (also known as product of SLC9A1, solute carrier family 9A, type 1). NHE-1 is ubiquitous, being expressed on the plasma membrane of virtually every mammalian cell. It mediates the 1:1 exchange of extracellular Na+ for intracellular H+, thereby maintaining intracellular pH. NHE-1 also plays cell-specific roles in cell volume maintenance, mitogenesis, cell-cycle regulation, apoptosis and a host of other cellular functions. NHE-1 has also been implicated in clinically relevant conditions such as hypertension, left ventricular hypertrophy, and ischemia-reperfusion injury. Despite its ubiquitous expression in mammalian cells and its potential clinical relevance, much remains to be learned regarding the molecular mechanisms through which this important protein is regulated. We seek a better understanding of the mechanisms through which NHE-1 is activated. We propose a model based on the idea that phosphorylation of NHE-1 and binding of CaM to NHE-1, disrupt electrostatic tethers that occlude the proton sensing and transport regions of NHE-1 in its basal state. Disruption of the tethers allows access of ambient protons to the proton sensing and transport regions of NHE-1, thus resulting in its activation. The activation is potentially facilitated by CA-II, which could couple to NHE-1 in a "metabolon", and which could gain access to the proton sensing and transport regions of NHE-1 by sliding into a pocket created when the electrostatic tethers are disrupted. This model will be tested in three specific aims: Aim #1: We will examine the role of Jak2-induced tyrosine phosphorylation of CaM in the activation of NHE-1. Aim #2: We will examine the role of carbonic anhydrase type 2 (CA-II) as a key regulatory protein that increases the activity of NHE-1. Aim #3: We will examine the role of electrostatic interactions involving the carboxyl terminus of NHE-1 in its activation. We believe that the proposal has been significantly improved by focusing on evaluating this potentially unifying mechanism of activation of NHE-1, which is a natural outgrowth of our previous work in this area, and which could serve as the foundation for the development of a molecular model for the activation of NHE- 1. This proposal could also lay the foundation for future structural experiments utilizing NMR and/or crystallography methods.